Development of Cell-Scaffold Construct for Cartilage Tissue Regeneration

Abstract

The treatment of cartilage tissue damage and /or diseases is a great challenge because of the limited self- repair ability of cartilage tissue and several limitations prevailed in current clinical methods. In this context, development of cartilage construct by tissue engineering technique using scaffold derived from suitable biopolymers and stem cell is the most promising approach. In this study, silk-fibroin/chitosan (SF/CS) and its composite scaffolds, as artificial extra cellular matrix (ECM), and umbilical cord blood (UCB) derived human mesenchymal stem cells (hMSCs) as potential cell source, were used for developing cartilage construct. Firstly, hMSCs were isolated from UCB as a potential stem cell source, cultured and characterised. Phase contrast microscopy showed fibroblast-like morphology thus confirmed the cultured cells are of mesenchymal in nature. Flow cytometry analysis revealed >95% expression of CD105, CD73, and CD90, with <2% for CD45, CD34, CD11b, CD79α and HLA-DR expression by hMSCs at 4th passage. In vitro differentiation potential of UCB-hMSCs into osteocytes, chondrocytes, and adipocytes, the characteristic mesenchymal lineages, was confirmed by histological analysis. UCB-hMSCs seeded on SF/CS scaffolds was cultured and differentiated under static and dynamic culture (spinner flask) conditions to generate cartilage construct. Constructs cultured in spinner flask bioreactor consisted of 62% live cells, and showed 543% more cell density at the core than the constructs obtained in static system. The progression of chondrogenic differentiation during 21 days of culture in chondrogenic media was assessed by glycosaminoglycans (GAG) synthesis, alcian blue staining for proteoglycan matrix, and immunofluorescence staining for collagen-II (Col II) and aggrecan (Acan). This was further confirmed by quantitative PCR (qPCR) analysis, showing low Col I expression and upregulated Sox9, matrilin 3 (MATN3), Col II and Acan genes. The results demonstrated superiority of culture condition in promoting hMSCs proliferation and ECM formation.
Secondly, effort has been given to improve the differentiation of hMSCs, thereby cartilage type ECM formation by using SF/CS scaffolds loaded with glucosamine (Gl) and chondroitin sulfate (Ch) individually. The Gl loaded SF/CS scaffolds (SF/CS-Gl) showed improved hMSCs attachment and viability (82.45±2.6% live cells) together with an increase in cell metabolic activity and proliferation than SF/CS scaffolds. The presence of Gl in the scaffold has enhanced the synthesis of cartilage-specific GAG with maintained chondrocyte phenotype and significant gene up-regulation. Similarly, the chondrogenic potential of SF/CS scaffolds was also enhanced by the incorporation of Ch into SF/CS scaffold (SF/CS-Ch). An improvement in chondrogenic differentiation of hMSCs was evident by higher GAG secretion (247±20 μg/mg) than SF/CS (83.2±7 μg/mg) and SF/CS-Gl (130±6 μg/mg) scaffolds. Though cell viability and proliferation were comparable to SF/CS-Gl scaffold, the expression of chondrogenic genes and matrix proteins were higher in SF/CS-Ch scaffolds implying the positive effect of Ch in supporting chondrogenic differentiation leading to the cartilage specific ECM formation.
Considering the beneficial effect of GI and Ch in combination, an effort was further given to investigate the chondrogenic differentiation of hMSCs over Gl and Ch loaded composite SF/CS scaffolds (SF/CS-Gl-Ch). Among the scaffolds with varied concentration of Ch (0.5-1.5 wt%), SF/CS-Gl loaded with 1.5% Ch showed superiority in promoting cell attachment, viability, and proliferation. The DNA content of hMSCs in SF/CS-Gl-Ch (342.2±4 ng/ml) was two times higher than SF/CS-Ch (163±5 ng/ml) and SF/CS-Gl (153±5.2 ng/ml) scaffolds. The proteoglycan matrix staining was more prominent in SF/CS-Gl-Ch scaffold with higher GAG synthesis of 383±7 μg/mg and fluorescence intensity of Col II and Acan. The cartilage specific ECM produced in this study is higher than the constructs developed in reported literature. The chondrocytes in SF/CS-Gl-Ch showed higher expression of Sox9, Col II and Acan genes representing its superior chondrogenic potential than other constructs developed in this study. Therefore, Gl and Ch loaded SF/CS scaffold is proven to be an excellent scaffold matrix possessing superior tailor properties that can offer enhanced chondrogenic differentiation of hMSCs, and thus cartilage-specific ECM formation in a dynamic bioreactor system. The generated cartilage construct may pave the way for cartilage tissue regeneration for future clinical application.